Ubiquitin-proteasome-rich cytoplasmic structures in neutrophils of patients with Shwachman-Diamond syndrome

Abstract

Background: Shwachman-Diamond syndrome is an autosomal recessive disorder in which severe bone marrow dysfunction causes neutropenia and an increased risk of leukemia. Recently, novel particulate cytoplasmic structures, rich in ubiquitinated and proteasomal proteins, have been detected in epithelial cells and neutrophils from patients with Helicobacter pylori gastritis and several epithelial neoplasms.

Design and methods: Blood neutrophils from 13 cases of Shwachman-Diamond syndrome - ten with and three without SBDS gene mutation - and ten controls were investigated by confocal microscopy and ultrastructural immunocytochemistry using antibodies against ubiquitinated proteins, proteasomes, p62 protein, and Helicobacter pylori VacA, urease and outer membrane proteins.

Results: Many extensively disseminated particulate cytoplasmic structures, accounting for 22.78 ± 5.57% (mean ± standard deviation) of the total cytoplasm, were found in blood neutrophils from mutated Shwachman-Diamond syndrome patients. The particulate cytoplasmic structures showed immunoreactivity for polyubiquitinated proteins and proteasomes, but no reactivity for Helicobacter pylori products, which are present in particulate cytoplasmic structures of Helicobacter pylori-positive gastritis. Neutrophils from patients with Shwachman-Diamond syndrome frequently showed p62-positive autophagic vacuoles and apoptotic changes in 5% of cells. No particulate cytoplasmic structures were observed in most control neutrophils; however, in a few cells from two cases we noted focal development of minute particulate cytoplasmic structures, accounting for 0.74 ± 0.56% of the total cytoplasm (P<0.001 versus particulate cytoplasmic structures from mutated Shwachman-Diamond syndrome patients). Neutrophils from non-mutated Shwachman-Diamond-syndrome-like patients resembled controls in two cases, and a third case showed particulate cytoplasmic structure patterns intermediate between those in controls and those in mutated Shwachman-Diamond syndrome patients.

Conclusions: Particulate cytoplasmic structures are a prominent feature of neutrophils from patients with Shwachman-Diamond syndrome. They may help us to understand the mechanism of granulocyte dysfunction and the neoplastic risk of the disease.

Figure 1.

(A) Electron microscopy (4,000x) of blood neutrophils from an SDS patient with SBDS gene mutation showing several PaCS scattered in the cytoplasm. The boxed area is enlarged in a1 (20,000x) to show PaCS-concentrated FK1 immunogold reactivity for polyubiquitinated proteins. In the inset (a2, 200,000x) from the same specimen as a1, the finely punctuate ultrastructure and barrel-like morphology of PaCS are shown at high resolution. Preferential PaCS reactivity of 20S (a3, 25,000x) and 19S (a4, 14,000x) proteasome antibodies is also seen. Black asterisk: PaCS; g: secretory granule. (B) PaCS-free control neutrophil (4,000x) enlarged in b1 (12,000x) to show thin areolae (white arrows) with preferential FK1 immunoreactivity. Scarce 20S proteasome immunoreactivity is seen in another section of the same control cell, with scattered areolae (b2, 12,000x). (C) Control neutrophil with focal development of small PaCS (boxed in C; 3,000x) enlarged in c1 (10,000x) and c2 (40,000x), with PaCS-concentrated FK1 immunogold. White asterisk: ribosomes. (D) One of the few neutrophils from an SDS-like syndrome without SBDS gene mutation showing PaCS. Note focal development of small PaCS, boxed in D (6,000x) and enlarged in d1 (35,000x) to recognize their particulate substructure and FK1 immunoreactivity. (E, 3,000x) A representative neutrophil from another SDS-like case shows on the left large, polarized PaCS, one of which is enlarged in e1 (30,000x) to illustrate its barrel-like particles and FK1 immunoreactivity; note PaCS-free cytoplasm on the right.

Figure 2.

Correlative confocal and electron microscopy of PaCS in SDS neutrophils. (A, 3,000x) Confocal microscopy of an SDS neutrophil immunostained with FK2 antibody against ubiquitinated proteins. Note intensely immunofluorescent areas which, when projected on a TEM micrograph at the same enlargement (A1, 3,000x), show substantial overlapping with PaCS (A2, 3,000x) as confirmed by the intense FK1 immunogold reactivity at higher enlargement (a3, 20,000x). (A4, 3,000x) 20S proteasome immunofluorescence of cytoplasmic areas in another neutrophil from the same SDS patient. (B and C) Autophagic vacuoles and PaCS in SDS neutrophils. Note in B (10,000x) the FK1 reactivity of PaCS, one of which (arrowhead) is apparently discharging its content into the vacuole, and in C (3,000x; enlarged in c1, 20,000x), the p62 reactivity of the vacuole and cytoplasm with, however, no reactivity of PaCS. (D–F, 3,000x) Three apoptotic neutrophils with typical chromatin condensation and segregation (target-type in E and crescentic in F) from SDS patients with SBDS gene mutations. In D, enlarged in d1 (10,000x), cytoplasmic homogenization with disappearance of organelles structure can be seen, whereas in E, enlarged in e1 (10,000x), secretory granules and some FK1 immunoreactivity are still recognizable. Note cytoplasmic vacuoles (of autophagic origin?) in all apoptotic cells. (G, 2,500x) Intraepithelial neutrophil in a gastric biopsy specimen from a patient with H. pylori gastritis, enlarged in g1 (36,000x) to show several PaCS intensely reactive for H. pylori urease. ep: epithelial cell; bm: basal membrane; sg: secretory granules; arrowheads: ribosomes.